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dictyNews Volume 18 Number 08
Dicty News
Electronic Edition
Volume 18, number 8
May 18, 2002
Please submit abstracts of your papers as soon as they have been
accepted for publication by sending them to dicty@northwestern.edu.
Back issues of Dicty-News, the Dicty Reference database and other useful
information is available at DictyBase--http://dictybase.org.
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Abstracts
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The Cdk5 homologue, Crp, regulates endocytosis and secretion in
Dictyostelium and is necessary for optimum growth and differentiation
Shiv K. Sharma1, Debra A. Brock2, Robin R. Ammann2 , Tiffany DeShazo3,
Meenal Khosla1, Richard H. Gomer2,3 and Gerald Weeks1
1Department of Microbiology and Immunology, The University of British
Columbia, Vancouver, BC V6T 1Z3 Canada
2Howard Hughes Medical Institute and
3Department of Biochemistry and Cell Biology,
MS-140, Rice University,
6100 S. Main Street,
Houston, TX 77005-1892
Developmental Biology, in press
Summary
Dictyostelium Crp is a member of the cyclin dependant kinase (Cdk)
family of proteins. It is most related in sequence to mammalian Cdk5, which
unlike other members of the family, has functions that are unrelated to the
cell cycle. In order to better understand the function of Crp in
Dictyostelium, we overexpressed a dominant negative form, Crp-D144N, under
the control of the actin 15 promoter. Cells overexpressing Crp-D144N
exhibit a reduced growth rate in suspension culture and reduced rates of
fluid-phase endocytosis and phagocytosis. There is no reduction in Cdc2
kinase activity in extracts from cells overexpressing Crp-D144N, suggesting
that the growth defect is not due to inhibition of Cdc2. In addition to
the growth defect, the act15::crp-D144N transformants aggregate at a slower
rate than wild-type cells and form large aggregation streams. These
eventually break up to form small aggregates and most of these do not
produce mature fruiting bodies. The aggregation defect is fully reversed
in the presence of wild-type cells but terminal differentiation is only
partially rescued. In act15::crp-D144N transformants, the countin component
of the counting factor, a secreted protein complex that regulates the
breakup of streams, mostly appears outside the cell as degradation products
and the reduced level of the intact protein may at least partially account
for the initial formation of the large aggregation streams. Our
observations indicate that Crp is important for both endocytosis and efflux
and that defects in these functions lead to reduced growth and aberrant
development.
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Clarke, M., Koehler, J., Arana, Q., Liu, T., Heuser, J., and Gerisch, G.
Dynamics of the vacuolar H+-ATPase in the contractile vacuole complex and the
endosomal pathway of Dictyostelium cells.
J. Cell Sci., in press.
ABSTRACT
The vacuolar H+-ATPase (V-ATPase) is a multi-subunit enzyme that plays
important roles in eukaryotic cells. In Dictyostelium, it is found
primarily in membranes of the contractile vacuole complex, where it
energizes fluid accumulation by this osmoregulatory organelle, and also in
membranes of endo-lysosomes, where it serves to acidify the endosomal lumen.
In the present study, a fusion was created between vatM, the gene encoding
the 100-kDa transmembrane subunit of the V-ATPase, and the gene encoding
Green Fluorescent Protein (GFP). When expressed in Dictyostelium cells,
this fusion protein VatM-GFP was correctly targeted to contractile vacuole
and endo-lysosomal membranes, and was competent to direct assembly of the
V-ATPase enzyme complex. Protease treatment of isolated endosomes indicated
that the GFP moiety, located on the C-terminus of VatM, was exposed to the
cytoplasmic side of the endosomal membrane rather than to the lumenal side.
VatM-GFP labeling of the contractile vacuole complex revealed clearly the
dynamics of this pleiomorphic vesiculo-tubular organelle. VatM-GFP labeling
of endosomes allowed direct visualization of the trafficking of vacuolar
proton pumps in this pathway, which appeared to be entirely independent
from the contractile vacuole membrane system. In cells whose endosomes
were pre-labeled with TRITC-dextran and then fed yeast particles, VatM-GFP
was delivered to newly formed yeast phagosomes with the same time course as
TRITC-dextran, consistent with transfer via a direct fusion of endosomes
with phagosomes. Several minutes were required before the intensity of the
VatM-GFP labeling of new phagosomes reached the level observed in older
phagosomes, suggesting that this fusion process was progressive and
continuous. VatM-GFP was retrieved from the phagosome membrane prior to
exocytosis of the indigestible remnants of the yeast particle. These data
suggest that vacuolar proton pumps are recycled by fusion of advanced with
newly-formed endosomes.
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A Talin Fragment as an Actin Trap Visualizing Actin Flow in Chemotaxis,
Endocytosis, and Cytokinesis
Igor Weber, Jens Niewoehner, Aiping Du, Ursula Roehrig, and Guenther Gerisch
Max-Planck-Institut fuer Biochemie, Martinsried, Germany
Cell Motility and the Cytoskeleton, in press.
ABSTRACT
A C-terminal 63 kDa fragment of talin A from Dictyostelium discoideum forms
a slowly dissociating complex with F-actin in vitro. This talin fragment
(TalC63) has been tagged with GFP and used as a trap for actin filaments
in chemotactic cell movement, endocytosis, and mitotic cell division.
TalC63 efficiently sequesters actin filaments in vivo. Its translocation
reflects the direction and efficiency of an actin flow. Along the body
of a migrating Dictyostelium cell, this flow is directed from the front
to the tail. If during chemotaxis one or two new fronts are induced, the
flow is always directed away from these fronts. The flow thus reflects
the re-programming of cell polarity in response to changing gradients of
chemoattractant. In endocytosis, the fluorescent complexes are translocated
to the base of a phagocytic or macropinocytic cup. During mitosis, the
complexes of F-actin with TalC63 accumulate within the midzone of anaphase
cells. If TalC63 is strongly expressed, the entire cleavage furrow is filled
out by sequestered actin filaments, and cytokinesis is severely impaired.
These cells are considered to mimic the phenotype of mutants deficient in
the shredding of actin filaments that normally occurs in the mid-zone of a
dividing cell.
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Tubular-Vesicular Transformation in the Contractile Vacuole System of
Dictyostelium. (Review)
Guenther Gerisch*, John Heuser#, and Margaret Clarke+
* Max-Planck-Institut fuer Biochemie, Martinsried, Germany
# Department of Cell Biology and Physiology, Washington University
School of Medicine, St. Louis, Missouri, USA
+ Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation,
Oklahoma City, Oklahoma, USA
Cell Biology International, in press.
ABSTRACT
The contractile vacuole complex of Dictyostelium is the paradigm of a membrane
system that undergoes tubular-vesicular transitions during its regular cycle
of activities. This system acts as an osmoregulatory organelle in freshwater
amoebae and protozoa. It collects fluid in a network of tubules and cisternae,
and pumps it out of the cell through transient pores in the plasma membrane.
Tubules and vacuoles are interconvertible. The tubular channels are associated
with the cortical actin network and are capable of moving and fusing. The
contractile vacuole complex is separate from vesicles of the endosomal pathway
and preserves its identity in a dispersed state during cell division. We
outline techniques to visualize the contractile vacuole system by electron
and light microscopy. Emphasis is placed on GFP-fusion proteins that allow
visualization of the dynamics of the contractile vacuole network in living
cells. Proteins that control activities of this specialized organelle in
Dictyostelium have been conserved during evolution and also regulate
membrane trafficking in man.
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[End Dicty News, volume 18, number 8]
